Project summary Microtubule (MT) motors such as kinesin-1 and dynein play critical roles in the cell by orchestrating the movements of proteins, organelles and cytoskeletal elements. In neurons, kinesin transports cargo towards the synapse while dynein takes it back to the cell body. This bidirectional transport is possible because the motors interact with the MT cytoskeleton in different ways. Kinesin walks towards the plus-end of MTs, while dynein walks towards the minus-end. For a cargo bound to both types of motors, as is often the case, the direction of travel will be dictated by which type of motor is more active. For example, if kinesin is more active than dynein, the cargo will move towards the synapse. By locally controlling the relative activity of these two motors, the cell can steer cargo to its proper destination. However, it is not well understood this regulation of motor activity occurs. Just as the MT surface dictates which direction the motors travel, it may also play a role in modulating their activity. In axons, motors move along MTs decorated with a gradient of the MT-associated protein Tau. It has been proposed that Tau spatially controls MT traffic by differentially regulating motor activity. In this model, the presence of Tau promotes the relative activity of one type of motor over the other. Using single-molecule imaging, the motility of the two motors on MTs coated with different concentrations of Tau will be measured. Then, the mechanism by which Tau affects motors will be determined using a variety of synthetic obstacles and Tau mutants. Finally, the study will be extended to multi-motor cargo bound to both kinesin and dynein and determine how Tau affects bidirectional transport. Together, these experiments will reveal the role that Tau plays in regulating bidirectional cargo traffic.